RNA interference (RNAi) signal can spread from the point where the double-stranded RNA (dsRNA) was initially applied to other cells or tissues. SID-related genes in Caenorhabditis elegans help in the spreading of this signal. However, the mechanisms of systemic RNAi are still not unveiled in insects. In this study, we cloned a full-length cDNA of sid-1-like gene, Pxylsid-1, from Plutella xylostella that contains 1,047 bp opening reading frame encoding a putative protein of 348 amino acids. This transcript is very much similar to the sil-1 in Bombyx mori (68.8%). The higher expression levels of Pxylsid-1 were found at the adult and fourth-instar stages compared to the second-instar stage with 21.48- and 10.36-fold increase, respectively. Its expression levels in different tissues were confirmed with the highest expression in the hemolymph, which showed 21.09-fold increase than the midgut; however it was lower in other tissues. The result of RNAi by feeding bacterially expressed dsRNA targeting Pxylace-1, which showed that the mRNA level of Pxylace-1 decreased by 34.52 and 64.04% after 36- and 72-h treatment, respectively. However, the mRNA level of Pxylsid-1 was not significantly induced when the Pxylace-1 was downregulated. Furthermore, we found that downregulation of Pxylsid-1 did not affect the RNAi effect of Pxylace-1. Hence, the Pxylsid-1 may not be involved in absorption of dsRNA from the midgut fluid. A further study is needed to uncover the function of Pxylsid-1.

While it has been well characterized that chemosensory receptors in guts of mammals have great influence on food preference, much remains elusive in insects. Insect chemosensory proteins (CSPs) are soluble proteins that could deliver chemicals to olfactory and gustatory receptors. Recent studies have identified a number of CSPs expressed in midgut in Lepidoptera insects, which started to reveal their roles in chemical recognition and stimulating appetite in midgut. In this study, we examined expression patterns in midgut of 21 Spodoptera litura CSPs (SlitCSPs) characterized from a previously reported transcriptome, and three CSPs were identified to be expressed highly in midgut. The orthologous relationships between midgut expressed CSPs in S. litura and those in Bombyx mori and Plutella xylostella also suggest a conserved pattern of CSP expression in midgut. We further demonstrated that the expression of midgut-CSPs may change in response to different host plants, and SlitCSPs could bind typical chemicals from host plant in vitro. Overall, our results suggested midgut expressed SlitCSPs may have functional roles, likely contributing to specialization and adaption to different ecosystems. Better knowledge of this critical component of the chemsensation signaling pathways in midguts may improve our understanding of food preference processes in a new perspective.

Our previous works confirmed that RNAi efficiency can be achieved by injection of dsRNA into the fourth-instar larva or pupa in Spodoptera litura. However, it has been considered that the RNAi efficiency is refractory in lepidopteran insects. In the present study, we focused on the RNAi machinery in S. litura by using an our previously constructed transcriptome (GenBank TSA: GBBY00000000) to detect genes predicted to be involved in the RNAi response, including the R2d2, Loqs, Ago1, Ago2, Dic1 and Dic2. By using quantitative PCR, we found that these genes were highly expressed in the development stage of adult and in the tissue of fat body. We also found three homologues of the sid1-like gene responsible for dsRNA transport inside the cell in the S. litura transcriptome. These genes were preferentially expressed in the pupal stage and in the tissue of midgut. Z-test of neutral evolution analysis showed that only between Sl-sid1 and Sl-sid3 have the probability of rejecting the null hypothesis of strict-neutrality (dN = dS, P< 0.05), indicating no evolutionary pressure between Sl-sid1 and Sl-sid3. Our data support that S. litura may have a conserved RNAi mechanism, but that different tissues and/or developmental stages may differ in the efficacy of the RNAi response.

Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications.

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